State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin, 150040, China.
Mol Biol Rep. 2013 Feb;40(2):1385-96. doi: 10.1007/s11033-012-2182-y. Epub 2012 Oct 18.
Plant NADP-malic enzyme (NADP-ME, EC 1.1.1.40) participates in a large number of metabolic pathways, but little is known about the NADP-ME family in woody plants or trees. Here, we characterized the tree Populus trichocarpa NADP-ME (PtNADP-ME) family and the properties of the family members. Five NADP-ME genes (PtNADP-ME1-PtNADP-ME5) were found in the genome of Populus. Semi-quantitative RT-PCR analysis show that the transcription levels of PtNADP-ME1 in lignified stems and roots are clearly higher than in other tissues, and PtNADP-ME2, PtNADP-ME3, PtNADP-ME4 and PtNADP-ME5 are broadly expressed in various tissues. PtNADP-ME gene expression was found to respond to salt and osmotic stresses, and NaCl salts upregulated the transcripts of putative plastidic ones (PtNADP-ME4 and PtNADP-ME5) significantly. Further, the NADP-ME activities of Populus seedlings increased at least two-fold under NaCl, mannitol and PEG treatments. Also, the expression of PtNADP-ME2 and PtNADP-ME3 increased during the course of leaf wounding. Each recombinant PtNADP-ME proteins were expressed and purified from Escherichia coli, respectively. Coomassie brilliant blue and NADP-ME activity staining on native polyacrylamide gels showed different oligomeric states of the recombinant PtNADP-MEs in vitro. Noticeably, the cytosolic PtNADP-ME2 aggregates as octamers and hexadecamers while the plastidic PtNADP-ME4 resembles hexamers and octamers. The four PtNADP-ME proteins except for PtNADP-ME1 have high activities on native polyacrylamide gels including different forms for PtNADP-ME2 (octamers and hexadecamers) or for PtNADP-ME4 (hexamers and octamers). High concentrations of NADP substrate decreased the activities of all PtNADP-MEs slightly, while the malate had no effect on them. The kinetic parameters (V (max), K (m), K (cat), and K (cat)/K (m)) of each isoforms were summarized. Our data show the different effects of metabolites (influx into tricarboxylic acid cycle or Calvin cycle) on the activity of the individual PtNADP-ME in vitro. According to phylogenetic analysis, five PtNADP-MEs are clustered into cytosolic dicot, plastidic dicot, and monocot and dicot cytosolic groups in a phylogenetic tree. These results suggest that woody Populus NADP-ME family have diverse properties, and possible roles are discussed.
植物 NADP-苹果酸酶（NADP-ME，EC 1.1.1.40）参与了大量的代谢途径，但关于木本植物或树木中的 NADP-ME 家族知之甚少。在这里，我们对杨树 NADP-ME（PtNADP-ME）家族及其家族成员的特性进行了描述。在杨树基因组中发现了五个 NADP-ME 基因（PtNADP-ME1-PtNADP-ME5）。半定量 RT-PCR 分析表明，木质化茎和根中 PtNADP-ME1 的转录水平明显高于其他组织，而 PtNADP-ME2、PtNADP-ME3、PtNADP-ME4 和 PtNADP-ME5 在各种组织中广泛表达。发现 PtNADP-ME 基因表达对盐和渗透胁迫有反应，NaCl 盐显著地上调了假定的质体（PtNADP-ME4 和 PtNADP-ME5）的转录物。此外，在 NaCl、甘露醇和 PEG 处理下，杨树幼苗的 NADP-ME 活性至少增加了两倍。此外，在叶片受伤过程中，PtNADP-ME2 和 PtNADP-ME3 的表达增加。分别从大肠杆菌中表达和纯化了每个重组 PtNADP-ME 蛋白。考马斯亮蓝和 NADP-ME 活性染色显示了重组 PtNADP-MEs 在体外的不同寡聚状态。值得注意的是，细胞质 PtNADP-ME2 聚集为八聚体和十六聚体，而质体 PtNADP-ME4 类似于六聚体和八聚体。除 PtNADP-ME1 外的四种 PtNADP-ME 蛋白在天然聚丙烯酰胺凝胶上具有高活性，包括 PtNADP-ME2（八聚体和十六聚体）或 PtNADP-ME4（六聚体和八聚体）的不同形式。高浓度的 NADP 底物略微降低了所有 PtNADP-ME 的活性，而苹果酸对它们没有影响。总结了每个同工酶的动力学参数（V（max）、K（m）、K（cat）和 K（cat）/K（m））。我们的数据表明，不同的代谢物（流入三羧酸循环或卡尔文循环）对个体 PtNADP-ME 在体外活性的不同影响。根据系统发育分析，五个 PtNADP-ME 在系统发育树中聚类为细胞质双子叶、质体双子叶和单子叶和双子叶细胞质组。这些结果表明，木本杨树 NADP-ME 家族具有多种特性，并对其可能的作用进行了讨论。
